| 1. |
- Alastair, Kerr, et al.
(författare)
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The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylasefunction regulating human fat cell metabolism
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pleiotropic function of long noncoding RNAs (lncRNAs) is well recognized,but their direct role in governingmetabolic homeostasis is less understood. Herein,we describe a human adipocyte-specific lncRNA, ADIPINT, that regulatespyruvate carboxylase (PC) an enzyme pivotal to energy metabolism. With a novelapproach, Targeted RNA-protein identification using Orthogonal Organic PhaseSeparation (TROOPS) and validation with electron microscopy, we show thatADIPINT binds to PC. ADIPINT knockdown alters the interactome anddecreases the mitochondrial abundance and enzymatic activty of PC. Decreases inADIPINT or PC expression reduce adipocyte lipid synthesis, breakdown and lipidcontent. In human white adipose tissue, ADIPINT expression is increased inobesity, linked to fat cell size, adipose insulin resistance and PC activity. Thus, weidentify ADIPINT as a regulator of lipid metabolism in human white adipocytes,which at least in part is mediated through its interaction with PC.
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| 2. |
- Arruda, Lucas C. M., et al.
(författare)
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A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo
- 2022
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 107:8, s. 1786-1795
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Tidskriftsartikel (refereegranskat)abstract
- Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34(+) blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34(+)CD38(-) phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34(+) hematopoietic stem cells from peripheral blood stem cell grafts and CD34(+) blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34(+) blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.
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| 3. |
- De Oliveira, Danilo Hirabae, et al.
(författare)
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Silk Assembly against Hydrophobic Surfaces?Modeling and Imaging of Formation of Nanofibrils
- 2023
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Ingår i: ACS Applied Bio Materials. - : AMER CHEMICAL SOC. - 2576-6422. ; 6:3, s. 1011-1018
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Tidskriftsartikel (refereegranskat)abstract
- A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, fi-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of similar to 7 nm and a width of similar to 11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.
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| 4. |
- de Rivero Vaccari, J. P., et al.
(författare)
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Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)
- 2023
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Ingår i: Translational Research. - : Elsevier BV. - 1931-5244 .- 1878-1810. ; 251, s. 27-40
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Tidskriftsartikel (refereegranskat)abstract
- Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1β release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.
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| 5. |
- Farsi, Zohreh, et al.
(författare)
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Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity
- 2018
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
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| 6. |
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| 7. |
- Gowrisankaran, Sindhuja, et al.
(författare)
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Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge
- 2020
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Ingår i: Journal of Molecular Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 0022-2836 .- 1089-8638. ; 432:4, s. 1235-1250
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Tidskriftsartikel (refereegranskat)abstract
- The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature-it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).
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| 8. |
- Kerr, A. G., et al.
(författare)
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The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.
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| 9. |
- Kerr, Alastair, et al.
(författare)
-
The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylase function regulating human fat cell metabolism
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pleiotropic function of long noncoding RNAs (lncRNAs) is well recognized, but their direct role in governing metabolic homeostasis is less understood. Herein, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase (PC) an enzyme pivotal to energy metabolism. With a novel approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation (TROOPS) and validation with electron microscopy, we show that ADIPINT binds to PC. ADIPINT knockdown alters the interactome and decreases the mitochondrial abundance and enzymatic activty of PC. Decreases in ADIPINT or PC expression reduce adipocyte lipid synthesis, breakdown and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity, linked to fat cell size, adipose insulin resistance and PC activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with PC.
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| 10. |
- Mim, Carsten, et al.
(författare)
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Structure versus function : Are new conformations of pannexin 1 yet to be resolved?
- 2021
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 153:5
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Tidskriftsartikel (refereegranskat)abstract
- Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.
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| 11. |
- Sporny, Michael, et al.
(författare)
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Structural basis for SARM1 inhibition and activation under energetic stress
- 2020
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 angstrom resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.
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| 12. |
- Sporny, Michael, et al.
(författare)
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Structural Evidence for an Octameric Ring Arrangement of SARM1
- 2019
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Ingår i: Journal of Molecular Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 0022-2836 .- 1089-8638. ; 431:19, s. 3591-3605
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Tidskriftsartikel (refereegranskat)abstract
- SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD + to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM(1-2) domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM(1-2) and found that it also forms a closed octameric ring in the crystal lattice. The SAM(1-2) ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1-2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development. Elsevier Ltd. All rights reserved.
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| 13. |
- Wang, Zuoneng, et al.
(författare)
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Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:12, s. 6177-6177
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Forskningsöversikt (övrigt vetenskapligt/konstnärligt)abstract
- Over the last few years, cryo electron microscopy has become the most important methodin structural biology. While 80% of deposited maps are from single particle analysis, electrontomography has grown to become the second most important method. In particular sub-tomogramaveraging has matured as a method, delivering structures between 2 and 5 Å from complexes in cellsas well as in vitro complexes. While this resolution range is not standard, novel developments pointtoward a promising future. Here, we provide a guide for the workflow from sample to structure togain insight into this emerging field.
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| 14. |
- Wang, Zuoneng, et al.
(författare)
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CryoEM reveals BIN1 (isoform 8) does not bind to single actinfilaments in vitro
- 2021
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Ingår i: microPublication biology. - : Caltech Library. - 2578-9430.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery thatbends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garneredattention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly. BIN1 (animportant protein in Alzheimer’s Disease, heart disease and cancer) is one of the few BAR proteins that bind to actindirectly. Here, we imaged a complex of BIN1 and actin with cryoEM. Our results reveal that BIN1 cannot be found onsingle actin filaments.
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| 15. |
- Wang, Zuoneng, 1991-, et al.
(författare)
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Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag
- 2022
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 189, s. 105992-105992
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at themembrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheralmembrane proteins remains challenging; mostly because there is no unified purification strategy forthese proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane.There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrialultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of themembrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure ofFAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domainproteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structuredetermination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To ourknowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Ourresults show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* isactive because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is apossible avenue to express and purify other challenging BAR domain proteins.
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| 16. |
- Wang, Zuoneng, 1991-
(författare)
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Structure studies of membrane associated proteins by transmission electron microscopy
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell membranes need to change their shapes during many cellular processeslike protein trafficking, cytokinesis and membrane homeostasis. The lattershuttles lipids, synthesized in the endoplasmic reticulum, to all membranouscompartments. Bin/Amphiphysin/Rvs (BAR) proteins are peripheralmembrane proteins (PMP) and play an important role in sculpturingmembranes and in the regulation of actin dynamics. Cryo-electronmicroscopy (cryoEM) has emerged as a powerful tool to visualize proteinsat the membrane interface. Here, we employed transmission electronmicroscopy and other biophysical methods to elucidate how BAR domainproteins steer processes at the membrane.In this work we studied the BAR protein bridging integrator 1 (BIN1), whichhas an established role in cancer, Alzheimer’s disease and skeletalmyopathies. To obtain information about BIN1’s interaction with themembrane in near native environments, we used artificial lipid systems suchas liposomes and lipids nanotubes.First, we have shown that electrostatic interactions are more important forBIN1 when binding to membranes with low curvature. At high curvature,binding is likely driven by non-polar interactions. The formation ofinvaginations (or tubules) is regulated by the composition of negativecharged lipids in membrane bilayer or electrostatic residues on the BARdomain. Therefore electrostatic interactions regulate recruitment andcrowding of BIN1; and consequently membrane deformation.Second, we clarified BIN1’s role in actin dynamics. CryoEM reveals that themuscular BIN1 isoform does not bind to single actin filaments, althoughBIN1 can be co-sedimented with actin after polymerization of actin. Thisimplies that BIN1 rather bundles actin than decorates single filaments.Third, we explored a strategy to purify an aggregation prone BAR protein.Aggregation is a property common in Peripheral Membrane Proteins. Thenovel NT* tag is derived from a spider silk protein and was reported to be apromising fusion tag for protein purification. We showed that the NT* tagimproves the solubility and reduces the aggregation of the BAR proteinFAM92A1. The activity of purified FAM92A1-NT* was verified bynegative stain EM.IIFourth, we were interested in the regulation of the lipid metabolism. PyruvateCarboxylase (PC) is a pivotal enzyme to generate lipid precursors. Cellbiological assays identified a long non-coding (lnc) RNA that regulates theactivity of PC. We studied the interaction between the lnc RNA and PC bybiophysical techniques. Size exclusion chromatography confirmed thepresence lncRNA-PC complex in vitro.
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